Survival of unfed Rhipicephalus appendiculatus and Rhipicephalus zambeziensis adults

Two ixodid tick species, Rhipicephalus appendiculatus (Neumann) and Rhipicephalus zambeziensis (Walker et al.) (Acari: Ixodidae), both originating from the southern province of Zambia, were used to study the survival time of adults at a range of different humidities and temperatures. In general, the experiment clearly demonstrates the different survival times of the two species in relation to the climatic conditions tested: survival of R. zambeziensis was better under more extreme conditions of temperature and humidity. These findings offer an explanation for the different distribution ranges of the two tick species. Rhipicephalus appendiculatus is more confined to cooler and wetter conditions, whereas R. zambeziensis is adapted to hotter and drier areas.     

A study of the diapausing behaviour of Rhipicephalus appendiculatus and R. zambeziensis under quasi-natural conditions in Zambia

Abstract. The diapausing behaviour of Rhipicephalus appendiculatus was studied under quasi-natural conditions in the Eastern Province of Zambia. Newly-moulted adults of ticks indigenous to the study area entered a behavioural diapause when exposed to daylengths below a critical photoperiod, provisionally a daylength between 11 h 20min and 11 h 45min. In the Eastern Province of Zambia the diapause was apparently not terminated by a long-day signal, but by a weakening of the photoperiodic maintenance of the diapause because of increasing age of the ticks. Adults of a reference stock indigenous to Kenya also entered a diapause when exposed to daylengths below the same threshold and maintained this diapause for the same length of time. Adults of a reference stock of Rhipicephalus zambeziensis indigenous to Zimbabwe did not enter a diapause when exposed to the same daylengths. The relevance of the findings is discussed in relation to the distributions of the two species.     

Molecular individuality and adaptation of the tick Rhipicephalus appendiculatus in changed feeding environments

The tick Rhipicephalus appendiculatus Neumann (Acari: Ixodidae) naturally infests many host species. However, the mechanisms that enable it to feed on such a wide range of hosts are unclear. One possibility is that a tick population maintains molecular (genotypic and/or phenotypic) diversity among individuals such that individuals vary in their competency in taking bloodmeals under different feeding conditions. As a first step in testing this hypothesis, we showed that the polymorphism of salivary gland proteins, previously demonstrated in unfed ticks, was maintained during feeding on guinea-pigs. We then compared feeding performance under standard laboratory rearing conditions: one instar (adults or nymphs) feeding on guinea-pigs, with three changed conditions: (1) two instars (adults and nymphs) feeding together on guinea-pigs; (2) one instar (adults or nymphs) feeding on hamsters; and (3) two instars (adults and nymphs) feeding together on hamsters. The mean engorged weight of adult females was significantly reduced under all changed conditions, indicating that most of the adult individuals were significantly challenged by the changed conditions. However, some individuals achieved successful engorgement, indicating competence to the changed condition, and demonstrating variation in adaptive ability among individuals. Engorged females produced egg masses positively correlated to the engorged weights. More interestingly, the correlation coefficient (R) increased when feeding condition was changed. This may lead to more efficient selection for population adaptation under the changed conditions. As the feeding success of ixodid ticks depends on the efficiency of the cocktail of immunomodulatory saliva, the relevance of the polymorphism of salivary gland proteins and host adaptation is discussed.     

Development of resistance in rabbits to immature stages of the Ixodid tick Rhipicephalus appendiculatus

A study of acquired resistance in rabbits to larvae and nymphs of Rhipicephalus appendiculatus Neumann showed a positive correlation between the levels of gamma globulin in the serum and the resistance developed by the host. A negative correlation between the weight of engorged ticks and gamma globulin levels was also demonstrated during this study. Repeated infestations with ticks evoked a typical antibody response to antigenic challenge. The levels of gamma globulin stabilized after three infestations and no further decrease in the weight of ticks was observed. The mechanism of the acquisition of resistance by the rabbits, the production of antibodies and the involvement of complement are discussed.     

Immune suppression induced by the brown ear tick Rhipicephalus appendiculatus Neumann, 1901

The brown ear tick Rhipicephalus appendiculatus suppressed in vivo and in vitro immune responses in the rabbit. Humoral responses were suppressed during infestation; however, suppression was transient and was unrelated to previous exposure of hosts to similar tick infestations. Immune suppression was caused by putative lymphocytotoxic factor(s) in tick salivary secretions as evidenced by in vitro lymphocytotoxicity assays. In an identical study of Rhipicephalus zambeziensis, a closely related rhipicephalid, the immune response was not suppressed, indicating that immune suppression is not common to the genus Rhipicephalus.     

Theileria parva: variation in the infection rate of the vector tick, Rhipicephalus appendiculatus

The variation in Theileria parva infection rates of experimental batches of adult Rhipicephalus appendiculatus ticks, used during the course of several years, was examined. It was found that considerable variation occurred, but that this could not always be correlated with the piroplasm parasitaemia in the cattle on which the ticks engorged as nymphs. Statistical analysis showed that the infection rate of ticks fed on cattle with a parasitaemia of 41–50 per cent was significantly higher than that of ticks fed on cattle with lower parasitaemias. A number of experiments were then carried out in which one or several factors of this aspect of the host-parasite relationship remained constant whilst others were altered. None of these factors was seen to play a major part in the variation. Finally, randomly selected groups of 10 ticks which had dropped engorged as nymphs from the same animal on the same day were examined. The variation observed even in these groups was so great that it was concluded that the infection rate could depend on a factor such as the juxtaposition of possibly-infected gut epithelial cells and developing salivary glands during the nymphal moult.     

In vitro growth of tick tissues (Rhipicephalus appendiculatus neumann, 1901)

• 1.1. The medium, in which initial outgrowths and long survival of several kinds of tick tissue cells occurred, contained Hanks' balanced salt solution, amino acids and vitamins of Eagle's basal medium together with 20% ox serum.
• 2.2. Explants, comprising body contents of nymphs and adult ticks (Rhipicephalus appendiculatus) were cultured in Leighton culture tubes.
• 3.3. Nymphs, dissected 4 to 7 days after engorgement, seemed to be most satisfactory for the preparation of tick tissue cultures.
• 4.4. Initial movements in tissues and outgrowth of cells frequently occurred within 24 hours in vitro.
• 5.5. Four cell types were recognized in the outgrowths. Mitotic changes in the outgrowths were also observed among all cell types.
• 6.6. Some of the explant tissues commonly survived in vitro over 60 days and tissue movements and cell outgrowths were observed for 173 and 175 days respectively.
• 7.7. Explant transfers (subculturing) were carried out with some success. In one instance outgrowths continued for 100 days.
     

Salivary fluid secretion in the ixodid tick Rhipicephalus appendiculatus is inhibited by Thogoto virus infection

Adult Rhipicephalus appendiculatus ticks, infectedwith Thogoto (THO) virus or control, were fed on guinea pigs and removed atintervals throughout the feeding cycle. Salivary fluid secretion was measuredbyan in vitro technique. The salivary glandsof infected, partially-fed ticks secreted fluid in vitro at about 75% the rateof controls, but the difference between infected and controls among engorgedticks was not statistically significant. Basal and DA-stimulated levels ofcyclic AMP (cAMP) were determined in isolated glands and were significantlyaffected by THO virus infection. The differences in secretory rate amongcontroland infected ticks could not be explained in terms of altered cAMP levels.Haemolymph volume was measured by a tracer-dilution technique using³H-inulin. The mean haemolymph volume for both THO-infected andcontrol groups was between 23–24% body weight throughout the feedingcycle, indicating that infection by this arbovirus did not influence salivaryfluid secretion via altered haemolymph volume. The mechanism by which THO virusaffects secretory activity of its tick vector remains unknown.     

Abiotic and biotic determinants of the seasonal dynamics of the tick Rhipicephalus appendiculatus in South Africa

Abstract. The creation of a generic population model for the tick Rhipicephalus appendiculatus requires a detailed, quantified understanding of the interactions of these ticks with their biotic and abiotic environment in the different parts of their range, from the tropical regions of East Africa to the temperate regions of South Africa. The much greater seasonal variation in climatic conditions, particularly temperature, further from the equator introduces variable development rates and diapause into the life cycle. Estimates of natural temperature-dependent interstadial development periods, derived from a combination of published laboratory and field data, were applied to published data on the seasonal abundance of R.appendiculatus on three farms in the Eastern Cape Province of South Africa. This enabled an assessment of which ticks of one stage give rise to which ticks of the next stage, from which (a) the onset and duration of diapause in unfed adults, and (b) seasonal interstadial mortality indices in the form of fc-values, could be estimated. The contribution of biotic (tick density) and abiotic (climatic) factors as predictors of mortality at each life stage was investigated by step-wise multiple regression. Density-independent mortality at the female-to-larval stage is correlated with geographically variable climatic factors, minimum temperature at two farms and minimum relative humidity at the third. The other two stages are governed by density-dependent mortality, which, it is argued, may be caused largely by the hosts' acquired resistance to ticks. As expected on theoretical grounds, this density dependence is weaker nearer to the edge of the tick's range and for the more vulnerable immature stages.     

Augmentation of host's naturally acquired immunity by solubilized membrane-bound midgut proteins of the tick Rhipicephalus appendiculatus.

Immune resistance of rabbits to the hard tick Rhipicephalus appendiculatus induced by combined tick infestations and immunization with solubilized midgut membrane proteins were compared with resistance due to 1-3 infestations or immunization alone. Results demonstrate that the level of exposure of rabbits to ticks used in the study does not significantly affect the immune expression resulting from immunization and that the latter augments the resistance due to infestation.     

Attempts to transmit Babesia (Nuttallia) microti in laboratory mice by the tick Rhipicephalus appendiculatus

Irvin A. D. and Brocklesby D. W. 1972. Attempts to transmit Babesia (Nuttallia) microti in laboratory mice by the tick Rhipicephalus appendiculatus. International Journal for Parasitology, 2, 99–103. The rodent piroplasm Babesia microti is easily maintained in laboratory mice by syringe passage, but experimental tick transmission has not been successfully achieved. Attempts were made to effect experimental transmission using a tick species (Rhipicephalus appendiculatus) readily maintained in the laboratory. Larval ticks fed on parasitaemic mice failed to transmit infection when ground up and inoculated into clean mice. Nymphal ticks from the same batches also failed to transmit infection when applied to mice. Larval ticks applied to a rabbit into which parasitaemic blood was inoculated failed to become infected, even though the rabbit's blood remained infective for 3 days on mechanical passage into clean mice. Piroplasms were detected in the gut of engorged larvae but not in ova laid by female ticks which had engorged on parasitaemic mice.     

Some factors controlling the stimulation of sporogony of Theileria parva in its tick vector Rhipicephalus appendiculatus

Young A. S., Leitch B. L. and Mutugi J. J. 1984. Some factors controlling the stimulation of sporogony of Theileria parva in its tick vector, Rhipicephalus appendiculatus. International Journal for Parasitology14: 97–102. The effect of various temperature treatments on the sporogony cycle of Theileria parva in the salivary glands of unfed adult Rhipicephalus appendiculatus ticks of various ages was investigated. It was found that ticks incubated at 28 or 37°C would develop sporozoites infective to cattle but never in such large numbers as in ticks fed on rabbits. Heat stimulation of sporogony was possible for isolates of T. parva with minimal laboratory handling. The age of the ticks incubated at 28 or 37°C was important since sporozoites could only be induced at the earliest on day 27 or 28 p.repl. (post-repletion) and at the latest by day 41 p.repl. The age of ticks fed on rabbits was not as important for the production of sporozoites.     

Rhipicephalus appendiculatus and R. zambeziensis (Acari: Ixodidae) from Zambia: a molecular reassessment of their species status and identification

The closely related species Rhipicephalus appendiculatus and R. zambeziensis co-occur in a wide area in Zambia. In this area, specimens of both species have been collected on the same individual host at the same time. In addition, specimens that are morphologically intermediate between R. appendiculatus and R. zambeziensis have been found in this area. These observations cast some doubt on the species status of R. appendiculatus and R. zambeziensis. Because the two taxa have varied influences on the epidemiology of East Coast fever (ECF), a cattle disease for which they act as vectors, it is essential that their taxonomic status is clarified and their identification is accurate. Therefore a phylogenetic analysis of nucleotide sequences of the ITS2 and a fragment of the mitochondrial 12S rDNA was performed to reassess the specific status of both taxa. This revealed two well supported clades coinciding with R. appendiculatus and R. zambeziensis as two separate species. In order to facilitate species identification a PCR-RFLP diagnostic assay was developed based on BauI digestion of the ITS2 gene. This assay produced clear diagnostic banding patterns for the two species and is applicable over a wide range of both species’ distribution. Nucleotide sequence data reported in this paper are available in the GenBank™ under the accession numbers DQ849203–DQ849274.     

East coast fever: 60Co-irradiation of Theileria parva in its tick vector, Rhipicephalus appendiculatus

Three experiments were carried out in which Theileria parva was irradiated in its tick vector, Rhipicephalus appendiculatus. In the first experiment, infected unfed adult ticks were irradiated at doubling doses from 4 to 32 krad. Some of the ticks were then fed for 4, 5, 6, 7 and 8 days on rabbits, and the parasites in their salivary glands examined. Five male and 5 female ticks from each irradiation dose were put onto each of a pair of susceptible cattle, whose reactions were recorded. Increasing doses of irradiation resulted in progressive destruction of the parasites. All cattle receiving ticks irradiated at doses up to and including 16 krad died of East Coast fever (ECF), and one of the cattle receiving ticks irradiated at 32 krad died.In the second experiment, recently engorged nymphs were irradiated at 1, 2 or 4 krad, and moulting nymphs at doses of 2, 4, 8, 12, 16 or 32 krad. The salivary glands of the resultant adult ticks were examined after the ticks had fed for 4, 5, 6, 7 or 8 days on rabbits. Engorged nymphs irradiated at 4 krad failed to moult, whilst moulting nymphs irradiated at 32 krad moulted but failed to attach to rabbits. Doses of irradiation survived by the ticks had no apparent morphological effect on the parasites they contained.In the third experiment, infected unfed adult ticks were irradiated at 0, 5, 10, 15, 20 25 30, 35, 40, 45, 50 or 60 krad. The ticks were fed on rabbits for 5, 6 or 7 days. Some of them were then examined morphologically, whilst others were ground in MEM/BPA and aliquots of the supernatant used to inoculate groups of 5 cattle. The reaction of these cattle, together with the morphological examination of the parasites, suggested that increasing doses of irradiation destroyed increasing numbers of parasites.     

Three serine proteinases from midguts of the hard tick Rhipicephalus appendiculatus; cDNA cloning and preliminary characterization

Rhipicephalus appendiculatus is one of the most economically important ticks distributed in south central and eastern Africa where little or no progress has been made on attempts to develop a vaccine. We have used a combination of RT-PCR, the 3 and 5rapid amplification of cDNA ends (RACE) to clone and sequence three cDNAs encoding full-length R. appendiculatus midgut serine proteinases (RAMSP). RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His⁵⁷ and Ser¹⁹⁵ conserved among most known serine proteinase-like genes . Northern blotting analysis of total RNA extracted from unfed and partially fed adult ticks revealed that mRNAs for RAMSP-1 and -2 were expressed only in partially fed ticks, while RAMSP-3 mRNA was not only expressed in both unfed and partially fed ticks, it was also up-regulated as tick feeding progressed. Expression analysis by RT-PCR revealed that RAMSP-3 was predominantly expressed in midguts when compared to salivary glands. For RAMSP-1 and -2, they were expressed at equivalent levels in both midguts and salivary glands. Based on key amino acid sequence features as well as similarity comparisons from the database, we speculated that polypeptides encoded by RAMPSP-1 to -3 are structurally more closely related to chymotrypsin- than trypsin-like serine proteinases. We have based our comments on the potential of serine proteinases as candidates for tick vaccines.     

Tick antigens recognized by serum from a guinea pig resistant to infestation with the tick Rhipicephalus appendiculatus

Immune resistance to infestation by an ixodid tick, Rhipicephalus appendiculatus, the vector of the cattle disease East Coast Fever, was induced in a guinea pig by repeated tick infestation. This resistance is expressed as the ability of the host to interfere with tick feeding. Resistance to ixodid tick feeding is an acquired response mediated by host antibody. We report the use of antibodies from a resistant host animal, in immunoblotting, to characterize the tick antigens recognized. The major tick antigens identified had molecular weights of 120,000, 94,000, 88,000, 77,000, 58,000, 46,000, 35,000, 31,000, 28,000, 25,000, 20,000 and 16,000. Most of these antigens were found in tick salivary glands. The presence and concentration of many tick salivary antigens appeared to vary with relation to the tick feeding cycle. Many of the antigens present in salivary glands were also detected in tick cement. Tick gut extract, although a poorer source of antigens, contained more of the 31,000 dalton antigen than salivary glands. Larval and nymphal tick extract lacked many of the antigens present in adult ticks. The data suggest that tick resistance is a complex phenomenon probably elicited by several different tick antigens.     

Feeding electrogram studies on the African cattle brown ear tick Rhipicephalus appendiculatus: evidence for an antifeeding effect of tick resistant serum

Abstract. Feeding behaviour of partially engorged Rhipicephalus appendiculatus (Neumann) (Acari: Ixodidae) on rabbit serum held in capillary tubes and placed over the tick mouthparts was studied using the feeding electrogram technique with simultaneous macro video photography. Correlation of electrical events with fluid movement in the vicinity of the tick's mouthparts and the capillary meniscus, permitted the characterization of an orderly sequence of signals, termed the 'Feeding Complex', associated with highest weight gains. This complex consisted of a 3–8 Hz fast-sucking waveform typically lasting 4–5 min, a sharp drop in potential at salivation, and rest lasting 1 or 2 min where no waveform or fluid movements occur. Very high impedence recordings from within the tick capitulum indicate that the fast-sucking waveform coincides with bursts of potentials corresponding to contraction of the pharyngeal dilator muscles, whereas during rest a tonic series of spikes signifies that the floor of the salivarium is actively lowered. Feeding electrograms of ticks fed on serum from tick-resistant rabbits showed significantly fewer feeding complexes. The weight gains achieved by these ticks were reduced correspondingly. This suggests that some of the humoral effectors of immunity have an antifeedant effect on this unusual parasite of rabbits.     

Evidence for a mounting sex pheromone in the brown ear tick Rhipicephalus appendiculatus,Neuman 1901 (Acari: Ixodidae)

The presence of a mounting sex pheromone was demonstrated on the surface of fed female Rhipicephalus appendiculatus. This pheromone, which is present on the female cuticle, allows the male to recognise the female. The pheromone was removed by cleaning the female in hexane, resulting in the loss of male mating behaviour in in vitro experiments. Male mating behaviour was resumed when extract made from fed female cuticle was replaced on cleaned females. When the extract was transferred to innanimate objects typical male mating behaviour was released. Preliminary chemical analyses indicated that the active component of the extract was contained in the sterol ester fraction of the extract.